This application is focused on dissection of the age- and caloric restriction-dependent changes in regulation of controlled proteolysis in mice tissues. The intracellular protein degradation is a vital part of all cellular metabolic processes. Proteins have to be destroyed or activated by proteolysis in a highly regulated manner to assure proper cell cycle rate, to promote immune response, to support signal transduction and to prevent the buildup of metabolic waste in the cell. The large enzymatic complex named proteasome alters its subunit composition and activities while the total cellular protein degradation declines with age. However, a mechanism of the global changes in proteolysis, a role of proteasome and other proteases in these changes, and the general role of proteasome in aging are largely unknown. We found that several large proteolytic complexes assist the proteasome in its metabolic duties. We also established that the activities of the complexes change with age in mice tissues. Moreover, we determined that changes in proteasomal subunit composition alter its function. On this basis we formulated our hypothesis that the cellular homeostasis requires a balanced collaboration between the proteasomes and several other cytosolic proteases, including the multicorn, a giant protease discovered in our laboratory, tripeptidyl peptidase II (TPPII), and leucine aminopeptidase (LAP). We postulate that the faulty interplay between the proteases significantly contributes to the age-related deregulation in the immune response. Signal transduction pathways, cell cycle abnormalities leading to cancer, and buildup of harmful protein waste. The ultimate goal will be to manipulate activities of the large cytosolic proteases in order to at least limit the age-dependent loss of equilibrium. Caloric restriction is the only currently available experimental paradigm that can alter the aging process. This intervention also modulates the age- related changes in proteasome structure and function. Therefore, testing the influence of caloric restriction on the equilibrium between the proteases will be of great importance. To set a solid basis for these studies we propose to: (I) establish how caloric restriction and age influence the contribution of large proteases: proteasome, TPPII, LAP and the multicorn to the total controlled proteolysis in liver, spleen, skeletal muscle and brain of the 6, 12, 18 and 24-months old C57BL/6 mice; (II) determine how they affect the subunit composition and cellular distribution of the proteasome and its regulatory complexes: 19S cap and 11S activator; (III) test how these changes in the cytosolic proteolytic activities influence fidelity of the proteolytic system using the processing of p105 to p50 in NFkB complex as an indicator, its robustness by testing the removal of oxidatively damaged proteins using oxidized myoglobin as model substrate, and effectiveness of metabolism of polyubiquitinylated substrates using fluorescently tagged ubiquitin. These studies will provide us with detailed pattern of age- dependent and caloric restriction-dependent changes in cellular nonlysosomal controlled proteolysis.